The actin cytoskeleton is essential in yeast and is composed of actin and numerous actin binding proteins. One actin binding protein, encoded by SAC6, is the yeast homolog of human fimbrin, an actin bundling protein (1). Sac6 protein is not essential for viability but is involved in many cytoskeletal functions. One common phenotype cytoskeletal mutants, including the sac6Δ, have is a defect in sporulation. Although this phenotype has been known for some time, the function of the actin cytoskeleton during sporulation is completely unknown. In order to determine the role of Sac6 protein and the actin cytoskeleton for sporulation, I accomplished the following: (1) I identified the terminal arrest point of the sac6Δ during sporulation as being immediately prior to spore wall formation, (2) By analyzing other mutants, I established that a primary function for the cytoskeleton during sporulation is for endocytosis, and (3) I identified an endocytic pathway in vegetative cells having different requirements for the actin cytoskeleton than the classical endocytic pathway. The events occurring during sporulation have been characterized. By using a number of different assays, I determined that the sac6Δ arrests late in the sporulation pathway. Different arrest points were seen depending on strain background used. However, in the SK1 background, a function for Sac6 protein in spore wall formation was identified. By examining other mutations defective for sporulation, I identified sla2Δ and chc1-521 as having sporulation defects similar to the sac6Δ. SLA2 encodes a cytoskeletal protein that has roles in endocytosis and CHC1 encodes the clathrin heavy chain that has roles in membrane trafficking, including endocytosis (2-4). Actin and Sac6 protein are also required for endocytosis (5). These data led to the model that a function of the actin cytoskeleton during sporulation is for endocytosis. An allelic series of actin mutations had previously been analyzed for ability to undergo receptor-mediated endocytosis (6). This data was compared with the sporulation ability of the actin mutations and a strong correlation was identified between these two phenotypes. I determined that endocytosis does occur throughout sporulation and that the sac6Δ has defects in endocytosis during sporulation. In order to better understand the role of endocytosis during sporulation, I analyzed the endocytosis of Ste6 protein. The half-life of this protein is known to be controlled by the endocytic machinery and it is endocytosed constitutively (7). The data obtained from this assay (although not informative with regards to sporulation) suggests that Ste6 protein has different requirements for the actin cytoskeleton than receptor-mediated endocytosis. All endocytosis appears to require the actin cytoskeleton, however this may be the first demonstration that multiple actin-dependent endocytosis pathways may exist in yeast.

Insulin action has been extensively studied although the exact mechanism by which the binding of this hormone to its receptor causes the observed effects is still obsure. A 90 kd membrane protein may be involved in this mechanism. An attempt was made to purify and make a monoclonal antibody to the 90 kd protein. Several purification methods were attempted and a 2-D electrophoretic procedure developed. Conventional hybridoma production methods were tried as well as a novel hybridoma procedure using selection of J11Dlo cells, culture in LPS/DxSO4, and electrofusion. The resulting monoclonal antibodies to the 90 kd protein were cross-reactive. Furthermore, the immunological results and the presence of the 90 kd protein in several mammalian species suggest that this protein may be evolutionarily conserved and may play a role in insulin action.

Amoeboid sperm from Ascaris contain an unusual cytoskeleton, composed of major sperm protein (MSP). In spermatozoa MSP filaments are organized into fiber complexes that extend from the leading edge to the base of the pseudopod and flow rearward at the same rate as the cell crawls forward. The tight coupling between cytoskeletal flow and motility indicates a key role for the MSP cytoskeleton in locomotion. I have studied both the regulation and structure of the MSP filament system in Ascaris sperm. Changes in intracellular pH (pH$\sb{\rm i}$) are involved in controlling the assembly status of MSP in vivo. Spermatozoa established a pseudopodial pH gradient with pH$\sb{\rm i}$ 0.15 units higher at the leading edge, where fiber complexes assemble, than at the base where disassembly occurs. Agents that abolished this gradient, such as weak acids, caused the cell to stop crawling and the fiber complexes to disassemble. The correlation between elevated pH$\sb{\rm i}$ and MSP assembly and low pH$\sb{\rm i}$ and depolymerization also was observed in developing sperm, indicating that pH regulation of the cytoskeleton also occurs during spermatogenesis. To understand cytoskeletal dynamics in greater detail, I studied the structure of MSP polymers. Self-association of monomers into helical assemblies is an intrinsic property of MSP. This feature is clearly displayed in the number of polymorphic forms MSP assumes in vivo and in vitro. We have characterized 4 distinct assemblies--subfilaments, filaments, macrofibers, and fiber complexes. Individual filaments formed either in vivo or in vitro are composed of two subfilaments wrapping around each other. These filaments coil around one another to form larger helical assemblies, macrofibers in vitro and fiber complexes in vivo. Filaments do not require accessory proteins to interact. Self-association of MSP filaments into / helical superstructures may have important implications for the role of MSP in sperm motility. This property could allow the filaments to form fiber complexes without requiring accessory proteins and, thus, create a simple framework to propel locomotion. / Source: Dissertation Abstracts International, Volume: 54-12, Section: B, page: 6023. / Major Professor: Thomas Michael Roberts. / Thesis (Ph.D.)--The Florida State University, 1993.

Two isozymes of creatine kinase (CK) were partially purified from isolated intestinal epithelial cells and identified as the non-muscle cytoplasmic isoform (B-CK) and the non-muscle mitochondrial isoform (Mi-CK). In intestinal epithelia, mitochondria (and Mi-CK) are excluded from the dense cytoskeletal web underlying the microvillar brush border. There are two myosin II subsets in this domain. The first cross-links the descending microvillar rootlets while the second is organized within a circumferential contractile ring. In glycerinated cells and isolated brush borders both interrootlet myosin solubilization and ring contraction were supported by the addition of creatine phosphate (Cr-P) to the endogenous B-CK-based ATP-regenerating system. When an exogenous ATP hydrolysis system was added (hexokinase and 25 mM glucose) both solubilization and contraction were supported in glycerinated cells, but only contraction was supported in isolated brush borders. Immunofluorescent imaging of B-CK revealed a marked loss in isolated brush borders compared to that of glycerinated cells, indicating that the interrootlet myosin-coupled B-CK subset had been extracted. These differential B-CK-myosin interactions were exploited to demonstrate ATP compartmentation between B-CK and ring myosin. Comparisons of epithelia from duodenum, jejunum, and ileum reveal that both contractile ring and interrootlet myosin-B-CK interactions are maintained despite the lower endogenous B-CK concentrations observed in the jejunum and ileum. / Source: Dissertation Abstracts International, Volume: 53-07, Section: B, page: 3260. / Major Professor: Thomas C. S. Keller, III. / Thesis (Ph.D.)--The Florida State University, 1992.

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